Primary Cells Research Articles

A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models.

Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. Here we report that SARS-CoV-2 replication is restricted at a post-entry step in a number of ACE2-positive airway-derived cell lines due to tonic activation of the cGAS-STING pathway mediated by mitochondrial DNA leakage and naturally occurring cGAS and STING variants. Genetic and pharmacological inhibition of the cGAS-STING and type I/III IFN pathways as well as ACE2 overexpression overcome these blocks. SARS-CoV-2 replication in STING knockout cell lines and primary airway cultures induces ISG expression but only in uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway in productively infected cells. Pharmacological inhibition of STING in primary airway cells enhances SARS-CoV-2 replication and reduces virus-induced innate immune activation. Together, our study highlights that tonic activation of the cGAS-STING and IFN pathways can impact SARS-CoV-2 cellular tropism in a manner dependent on ACE2 expression levels.

Airway epithelial cell response to RSV is mostly impaired in goblet and multiciliated cells in asthma

BackgroundIn patients with asthma, respiratory syncytial virus (RSV) infections can cause disease exacerbation by infecting the epithelial layer of the airways, inducing subsequent immune response. The type I interferon antiviral...

Effects of neratinib on angiogenesis and the early stage of the embryo using chicken embryo as a model.

Angiogenesis is the process of forming new blood capillaries from pre-existing vessels. Even though it is essential during normal development, it plays a major role in cancer progression. Neratinib is a pan-human epidermal growth factor receptor (HER) inhibitor that has recently been approved for the treatment of HER2-positive breast cancer. However, its effects on angiogenesis and embryogenesis remain unknown. This study examined the antiangiogenic effects of neratinib using the chorioallantoic membrane (CAM) of chicken embryos. We also evaluated neratinib's toxicity during the early stages of normal development using the chicken embryos, primary embryonic fibroblasts (EFBs), and human umbilical vein endothelial cells (HUVEC). Our findings revealed that neratinib significantly inhibited the CAM angiogenesis compared to controls by reducing vessel percentage area and the average vessel length. Furthermore, neratinib downregulated vascular endothelial growth factor (VEGF), a key mediator of angiogenesis. At lower concentrations, neratinib was well-tolerated during early stages of normal development. Additionally, EFBs treated with neratinib showed no morphological or viability changes when compared to controls. However, at the highest concentration tested, neratinib treatment reduced HUVEC cell viability. This effect may be associated with the dysregulation of key apoptotic genes, including caspase-3, caspase-8, caspase-9, and the B-cell lymphoma 2 (Bcl2) gene. Our findings indicate a novel potential application of neratinib as an antiangiogenic agent, exhibiting tolerable toxicity in the early stages of embryogenesis.

Human Chorionic Gonadotropin Regulates the Smad Signaling Pathway by Antagonizing TGF-β in Giant Cell Tumor of Bone.

Giant cell tumor of bone (GCTB) is a locally aggressive bone tumour aggravated by stromal cell proliferation and metastasis. We investigated the mechanism of action of human chorionic gonadotropin (HCG) in mediating GCTB proliferation and invasion. The expression of HCG was quantified using quantitative real-time PCR. After the primary stromal cells were isolated and identified, the function of HCG in GCTB was estimated using the cell counting kit-8, flow cytometry, scratch experiment, transwell assay, Western blot, and immunofluorescence. Moreover, the mechanism of HCG was assessed through western blotting. HCG expression was decreased in clinical tissue samples from patients with GCTB. We validated that HCG repressed stromal cell proliferation, migration, invasion, autophagy, and epithelial- mesenchymal transition (EMT) and promoted cell apoptosis in GCTB. We also verified that HCG repressed the autophagy and EMT of stromal cells through the Smad signaling axis in GCTB. HCG inhibited the transduction of the Smad signaling pathway by restraining the binding of the TGF-β II receptor to ligand Activin A. HCG restrained the Smad signaling pathway by antagonizing TGF-β signaling in GCTB. HCG may serve as a useful patent to treat GCTB.

Corydaline alleviates Parkinson's disease by regulating autophagy and GSK-3β phosphorylation.

Jitai tablet, a traditional Chinese medicine, has a neuroprotective effect on 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mice. As one of the main active ingredients in the Jitai tablet, corydaline (Cory) has analgesic and anti-allergic effects, but it has not been studied in PD. Here, we investigated the role and mechanism of Cory in PD. The PD model was induced by MPTP. Cell viability was measured by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide assay. The Pole test and traction test were performed to detect the behaviors of mice. The expression of tyrosine hydroxylase (Th) was detected by immunohistochemistry and Western blot. Immunofluorescence staining, monodansylcadaverine staining, and Western blot were conducted to assess autophagy. A lactic dehydrogenase release assay was used to detect cytotoxicity. Network pharmacology was used to screen the targets. There existed cytotoxicity when the concentration of Cory reached 40μg/mL. Cory (not exceeding 20μg/mL) could alleviate MPTP-induced cell damage. In vivo experiments indicated that Cory could improve the motor coordination of mice with PD. Besides, Cory could increase LC3-II/LC3-I levels both in vivo and in vitro. In addition, the Th levels reduced in the striatum and middle brain tissues of Parkinson's mice were recovered by Cory injection. We also found that Cory decreased the phosphorylation of glucogen synthase kinase-3 beta (GSK-3β) at Tyr216 and increased the phosphorylation of GSK-3β at Ser9 not only in primary neurons and SH-SY5Y cells but also in the striatum and middle brain tissues. Furthermore, Cory increased LC3-II/LC3-I levels and decreased p62 levels by regulating GSK-3β. Cory enhanced autophagy, attenuated MPTP-induced cytotoxicity, and alleviated PD partly through the regulation of GSK-3β phosphorylation.

An Innovative Approach to Assess the Potentiality of Using Activated Carbon and Rice Husk Ash in Aluminum-Air Battery

Aluminum-air (Al-air) cells have the potential to become vital in energy storage applications in the future because of their high energy density, which is even higher than that of commonly used lithium-ion batteries. However, it is not used widely because the cost of air cathode catalysts and metal anode is high. However, suppose the catalysts are replaced with activated carbon or rice husk ash as an alternative and recycled aluminum foil as an anode. In that case, the production cost might be feasible for the vast use of this type of cell. This study's main objective is to utilize some commonly available material in fabricating an Al-air battery suitable for small and day-to-day usage, reducing production costs and limitations. In this paper, a focused analysis was made on the feasibility of using an activated carbon and rice husk mixture as an air cathode catalyst for an Al-air cell, and the observations were interesting. About 11 samples of a mixture of rice husk ash (RHA) and activated carbon (AC) in different ratios have been made to find the best results from 0.68-0.72 V, which increases by 8-20%, measuring each sample after 3 days. In this study, another attempt was made to replace the graphite cathode of a dry cell with a mixture of AC and RHA. Voltage drop is quite negligible for the mixture of 10% RHA. The resulting voltage is similar to the new 100% activated carbon battery as a cathode. If considering the environmental effect, using recycled activated carbon and rice husk ash will decrease pollution and open a new door to apply in primary cells.

Akt-driven TGF-β and DKK1 Secretion Impairs F508del CF Airway Epithelium Polarity.

Epithelial polarity is fundamental in maintaining barrier integrity and tissue protection. In cystic fibrosis (CF), apicobasal polarity of the airway epithelium is altered, resulting in increased apical fibronectin deposition and enhanced susceptibility to bacterial infections. Here, we evaluated the effect of highly effective modulator treatment (HEMT) on fibronectin apical deposition and investigated the intracellular mechanisms triggering the defect in polarity of the CF airway epithelium. To this end, primary cultures of CF (F508del variant) human airway epithelial cells (HAECs) and a HAEC line, Calu-3, knocked-down (KD) for CFTR (CFTR KD) were compared to control counterparts, grown at an air-liquid interface (ALI). We show that CFTR mutation in primary HAECs and CFTR KD cells promote the overexpression and over-secretion of TGF-β1 and DKK1 when cultured at ALI. These dynamic changes result in hyperactivation of the TGF-β pathway and inhibition of the Wnt pathway through degradation of β-catenin leading to imbalanced proliferation and polarization. The abnormal interplay between TGF-β and Wnt signaling pathways is reinforced by aberrant Akt signaling. Pharmacological manipulation of TGF-β, Wnt, and Akt pathways restored polarization of the F508del CF epithelium, a correction that was not achieved by HEMT. Our data shed new insights into the signaling pathways that fine-tune apicobasal polarization in primary airway epithelial cells and may provide an explanation to the mitigated efficacy of HEMT on lung infection in people with CF.

Abstract 5895: N17350 kills cancer cells, spares immune cells, and regresses CDX tumors from chemotherapy naive and experienced patients

Abstract Background. While multiple studies have demonstrated improved clinical outcomes in patients receiving concurrent chemotherapy and immunotherapy, clinical synergy may be limited by the destructive effects of DNA-damaging chemotherapy on non-cancer cells, including key constituents of anti-tumor immunity (e.g. mature T cells). Furthermore, when first-line chemotherapy fails, subsequent lines of chemotherapy produce lower response rates. Thus, new approaches that eradicate cancer cells while preserving immune cells may be required to more efficiently induce anti-tumor immunity as monotherapy or in combination with immunotherapy. Neutrophil elastase (ELANE), a neutrophil-derived serine protease, kills a wide range of cancer cells without harming immune cells through a unique mechanism involving histone H1 and the death domain of CD95. Here, we compared N17350, an optimized biologic leveraging the ELANE pathway, and chemotherapies for their ability to kill cancer cells and preserve immune cells from primary tumors of ovarian cancer (OvCa) patients and evaluated efficacy in OvCa patient cell-derived xenografts (CDXs) and a 4T1 model of metastatic breast cancer. Methods. For in vitro studies, cancer cells and CD45+ immune cells were isolated from primary tumors of chemotherapy naïve and experienced OvCa patients. Cells were treated with N17350, oxaliplatin, or doxorubicin 24h post-isolation, and viability was quantified by calcein-AM. For in vivo studies, primary OvCa cells were injected into nude mice to create CDX models. Mice were treated with 2 doses of N17350 (400μg/100mm3, intratumoral) or carboplatin (100mg/kg, intraperitoneal), and effects on tumor growth were quantified. In 4T1 model, N17350 was compared to oxaliplatin, alone or in combination with anti-CTLA4, and effects on tumor growth and immune profile were assessed. Results. N17350 killed primary cancer cells from all OvCa patients tested but was well tolerated by CD45+ tumor immune cells from the same patients. In contrast, doxorubicin and oxaliplatin showed similar toxicity to both cell types. N17350 killed cancer cells from chemotherapy naïve and experienced patients with equal efficacy, while doxorubicin and oxaliplatin showed less efficacy in patients previously treated with chemotherapy. N17350 rapidly regressed tumors in all OvCa CDX models and exhibited markedly improved efficacy over carboplatin. In the 4T1 model, N17350 outperformed oxaliplatin in terms of regressing tumors, elevating tumor immune cells, and combining with anti-CTLA4. Conclusions. Our data demonstrate that N17350 kills OvCa tumors in vitro and in vivo while sparing CD45+ immune cells in both chemotherapy naïve and experienced patients. Together with previous data showing that N17350 induces immunogenic tumor cell death, these findings suggest that N17350 could improve clinical outcomes compared to chemoimmunotherapy. Citation Format: Chang Cui, Ravindra Gujar, Christine Lee, Maria Fumagalli, Nicole Martinez, Hannah Liu, Nicole Grigaitis, Sonia Feau, Afshin Bahador, Alain P. Algazi, Lev Becker. N17350 kills cancer cells, spares immune cells, and regresses CDX tumors from chemotherapy naive and experienced patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5895.

Abstract 1338: Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells

Abstract Human leukocyte antigen-G (HLA-G) suppresses lymphoid and myeloid immune systems by binding ILT2 and ILT4 respectively and activating ITIM signaling. HLA-G is abundantly expressed in placental cytotrophoblasts to block fetal rejection. HLA-G is not expressed in most normal cells but is derepressed in in about 50% of malignancies. We engineered ILT2 and ILT4 to produce activating signals as Chimeric ILT Receptors or CIR. Preliminary results indicated robust killing of Acute Myeloid Leukemia (AML) cells by Natural Killer (NK) cells expressing either ILT2 or ILT4 CIRs. Here, we evaluated the effect of alteration of activating domains in the CIR constructs to produce signals that mimic the effect of IL-18 signaling in CIR-NK cells to enhance cytotoxicity, NK cell growth and persistence. Further, HLA-G expression and CIR-NK cell targeting of primary human cell types were rigorously screened. Activated NK cells derived from peripheral blood (2 to 5 donors) were transduced with γ-retroviruses directing expression of CIR proteins (CIR.X.Y or CIR.Y.X) where X stimulates cytotoxicity through CD3ζ, DAP10 or DAP12, and Y directs coactivation through 4-1BB, fusion of the MyD88 death domain or recruitment of endogenous MyD88 with receptor-derived TIR domains and soluble IL-15. CIR-NK cells were cocultured with GFPffluc-expressing AML and solid tumor target cell lines or normal primary human cells. NK cell proliferation and cytotoxicity of CIR-NK cells was monitored by Incucyte microscopy and supernatants were analyzed for cytokine release by ELISA. At low E:T (1:20 - 1:40) in 7-day cocultures, mock-transduced NK cells displayed innate killing activity against HLA-G+ Molm13 and Kasumi1 AML cells that was augmented by CIR constructs containing direct fusions of MyD88 and DAP10 or DAP12 (mock = 2.06E7 ± 4.53E6 vs. CIR-ILT4.MyD88.DAP10 = 1.03E7 ± 5.46E6) with 7-fold elevation of IFN-γ production. CIR constructs that recruited endogenous MyD88 signaling through the TLR2 TIR domain (4-1BB.DAP10.TLR2) also exhibited enhanced anti-HLA-G activity (9.13E6 ± 5.33E6). Against HLA-G+ HT-1376 bladder cancer cells (E:T = 1:10-1:20), CIR-NK cells also displayed enhanced tumor cell killing with enhanced NK cell growth. Conversely, CIR-enhanced killing was not observed against HLA-G− HCT-116 colorectal cancer cells. The persistence of CIR-NK cell potency after 4 weeks of expansion varied markedly with different activation domains employed. When screened against primary human cells (cardiomyocytes, hepatocytes, hepatic endothelial cells, corneal, colon and lung epithelial cells), no CIR-specific targeting was observed at high E:T ratios (1:1 and 1:5). In conclusion, CIR-NK cells exhibit potent and specific anti-tumor activity against HLA-G+ solid and leukemia cells. Alteration of CIR-NK cell intracellular signaling can optimize the potency and persistence of NK cell anti-tumor activity. Citation Format: MyLinh Duong, Jihyun Park, Raphel Ognar, Simon Wain-Hobson, Henri Bayle. Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1338.

Abstract 279: Iatrogenic immunosuppression emplaces tacrolimus to drive squamous skin cancer progression and influence the microenvironment

Abstract One in thirteen organ transplant recipients (OTRs) develop post-transplant cutaneous squamous cell carcinoma (cSCC), leading to significant morbidity upon disease progression. Although chronic ultraviolet (UV) exposure primarily underlies keratinocyte carcinogenesis, long-term immunosuppression is a critical etiologic risk factor due to reduced systemic surveillance of dysplasia. Calcineurin inhibitors such as tacrolimus are commonly administered to suppress the immune system by blocking T-cell proliferation and preventing donor organ rejection. Contrarily, studies have evinced the off-target effects of immunosuppressives, namely azathioprine and cyclosporine, but not tacrolimus — the most widely used in all immunosuppression. In addition, the preclinical research literature has overlooked the contribution of stromal fibroblasts in the cSCC tumor microenvironment (TME). Hence, we hypothesize that tacrolimus exerts direct carcinogenic effects on keratinocytes and fibroblasts in the skin. We further postulate that tacrolimus enables a tumor-permissive microenvironment by activating dermal fibroblasts to cause aggressive and invasive keratinocyte carcinoma in OTRs. To examine this cellular crosstalk phenomenon, we used human keratinocytes, primary dermal fibroblasts and cSCC cells in mono- and co-cultures to mimic cutaneous and tumor complexities. Next, we assessed the molecular mechanisms utilized by tacrolimus to potentiate tumorigenesis pertinent to cell survival, proliferation and invasion. Herein, we show that tacrolimus promotes cell survival dose-dependently in cSCC cells. We also found that a fibroblast-cSCC spheroid co-culture significantly enhances cell invasion. In a wound-healing migration assay, tacrolimus increased the migratory capacity of normal keratinocytes and cSCC cells. However, cell proliferation remains nebulous due to cyclical dose-response effects in vitro. We then queried whether cell proliferation is affected in vivo using immunodeficient mice, and similar results were observed. We confirmed via immunoblotting that tacrolimus induced elevated expression of proliferation markers at distinct drug concentrations. Altogether, our findings illuminate keratinocyte-stroma interactions and particularly isolate the contributory role of tacrolimus in non-immune mediated cSCC pathogenesis. This knowledge suggests newer immunosuppressives that do not inhibit calcineurin activity may help relieve cutaneous malignancies in transplant recipients. Citation Format: Edwin Kumah, Yash Chhabra, Vania Wang, Agrani Dixit, Ashani Weeraratna, Kristin Bibee. Iatrogenic immunosuppression emplaces tacrolimus to drive squamous skin cancer progression and influence the microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 279.

Abstract 5898: Inhibition of the LONP1 ATPase domain induces mitochondrial proteotoxic stress and is cytotoxic to acute myeloid leukemia cells

Abstract Compared to normal hematopoietic cells, AML cells exhibit a heightened reliance on mitochondrial metabolism for survival and proliferation. To support this unique phenotype, AML cells increase import of nuclear-encoded mitochondrial proteins which must be properly folded by mitochondrial chaperones, proteases, and heat shock proteins. Failure to properly fold these precursors leads to protein aggregation, mitochondrial dysfunction, and cell death. To evaluate the reliance of AML cells on maintaining mitochondrial proteostasis, we assessed gene dependency datasets (eg: depmap.org) and identified the mitochondrial protease LONP1, as the top essential gene. LONP1 is a matrix-localized nuclear-encoded AAA+ protease. Proteins are unfolded by its ATPase domain and degraded by its serine-catalyzed proteolytic domain. Compared to hematopoietic cells, LONP1 mRNA was overexpressed in AML across three publicly available datasets. By immunoblotting, LONP1 protein was increased in 16/30 primary AML samples compared to bulk (n=8) and CD34+ blood cells (n=3). LONP1 genetic depletion reduced the growth and viability of OCI-AML2, OCI-M2, NB4, and TEX leukemia cells as well as primary AML cells. LONP1 depletion increased aggregated mitochondrial proteins and reactive oxygen species (ROS) production while reducing mitochondrial respiration and membrane potential. To determine the domain of LONP1 that was necessary for mitochondrial protein solubility, mitochondrial function, and AML survival, we over-expressed wild type, ATPase dead (E591A), or proteolytically dead (S855A) LONP1 cDNA in OCI-AML2 cells and knocked down endogenous LONP1 with shRNA targeting the 3’UTR of the endogenous gene. Wild type or proteolytically dead (S855A) but not the ATPase mutant (E591A) rescued mitochondrial protein solubility, mitochondrial respiration, ROS generation, membrane potential, and cell viability. Thus, the ATPase domain but not the proteolytic activity of LONP1 is necessary for mitochondrial proteostasis. Bardoxolone methyl (CDDO-Me) is a synthetic triterpenoid that allosterically inhibits the LONP1 ATPase site. CDDO-Me killed OCI-AML2 and NB4 cells with IC50 values of 178.5±29.7 and 156.5±39.7 nM, respectively. CDDO-Me (200 nM) killed 3 out of 4 primary AML samples with high LONP1 expression but none of the 10 primary AML samples with low LONP1. Likewise, CDDO-Me induced mitochondrial protein aggregation in AML cell lines and primary AML patient samples with high LONP1 expression but not AML cells or primary AML patient samples with low LONP1. In summary, LONP1 is over-expressed in a subset of AML cells and primary samples where it maintains mitochondrial protein solubility. Selective inhibition of the LONP1 ATPase domain leads to mitochondrial protein aggregation and AML cell death, representing a novel therapeutic strategy for AML. Citation Format: Matthew Tcheng, Marcela Gronda, Rose Hurren, Lan Xin Zhang, Chaitra Sarathy, Yongran Yan, Andrea Arruda, Mark D. Minden, Aaron D. Schimmer. Inhibition of the LONP1 ATPase domain induces mitochondrial proteotoxic stress and is cytotoxic to acute myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5898.

Abstract 4259: A novel human model of lung squamous cell carcinoma to deconvolve the cell-intrinsic effects of key dysregulated pathways

Abstract Tractable, patient relevant models are needed to investigate the essential components of normal cell transformation. We developed a unique in vitro model of early lung squamous cell carcinoma (LUSC) genetic alterations and driver pathway dysregulation using genetically modified primary human bronchial basal cells (hBCs) from three independent donors. The co-operation of ubiquitous and early LUSC alterations (TP53 and CDKN2A loss) and commonly deregulated pathways including squamous differentiation (SOX2), PI3K signaling (PTEN) and the oxidative stress response (KEAP1) was investigated using a combinatorial approach, culminating in eight unique hBC mutants harboring cumulative pathway alterations in a TP53-/-/CDKN2A-/- background. Phenotypes indicative of mutant hBC transformation were investigated using proliferation assays, organotypic cultures and anchorage independent growth assays. Organotypic cultures revealed LUSC pathway alterations to drive dramatic changes in airway histology, ranging from a normal epithelium to severe dysplasia. Furthermore, these analyses confirmed the role of SOX2-overexpression in initiating transformation by inducing squamous cell commitment and anchorage independent growth. We also identified a co-operation of SOX2-overexpression with the oxidative stress response and PI3K pathways to drive more advanced lesions in-vivo and in-vitro. Importantly, we were able to map changes in the expression of immunomodulatory factors with the pathway dysregulation. We demonstrated that this strategy constitute an alternative and patient-relevant system to model LUSC and identify genotype-phenotype correlations of clinical relevance. Citation Format: Julia Ogden, Robert Sellers, Anthony Oojageer, Sudhakar Sahoo, Anshuman Chaturvedi, Carlos Lopez-Garcia. A novel human model of lung squamous cell carcinoma to deconvolve the cell-intrinsic effects of key dysregulated pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4259.

Abstract 618: Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia

Abstract GCN2 (EIF2AK4) is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR), an adaptive cellular program triggered primarily by amino acid scarcity. Active GCN2 phosphorylates the translation initiation factor eIF2α, resulting in the attenuation of global protein synthesis and a largely ATF4-driven program of resource enhancement. GCN2 promotes cancer cell survival under conditions of microenvironmental or drug-induced intracellular nutrient scarcity. Here, we describe the preclinical development of APL-030, a novel and selective ATP-competitive inhibitor of GCN2. APL-030 was confirmed as a potent inhibitor of GCN2 in an on-target biochemical kinase assay with a Ki of 4.4nM. In a cell-based assay APL-030 decreased eIF2α phosphorylation in a dose-dependent manner via direct GCN2 inhibition with an IC50 of 50.8nM. In acute myeloid leukemia (AML) cell lines GCN2 inhibition resulted in a decrease in gene expression of effector genes CHAC1 and DDIT3. Moreover, treatment with APL-030 dose-dependently decreased cell viability in multiple AML cell lines, which was accompanied by an increase in caspase 3/7 activity. Bulk mRNA sequencing of MOLM-13 AML cells showed that APL-030 inhibited the extensive transcriptome changes triggered by glutamine depletion, in line with a high degree of selectivity of APL-030 for GCN2-mediated ISR signaling. In cells grown in rich medium, APL-030 triggered a largely ISR-independent transcriptome response that was dominated by the enrichment of pathways related to cell cycle as well as RNA and protein metabolism. In vivo, APL-030 treatment of an AML xenograft animal model completely inhibited tumor growth and prolonged survival time. Inhibition of AML tumor growth was shown to be a consequence of decreased cell viability and induction of caspase-dependent apoptosis. APL-030 was also tested on primary diagnostic AML cells, which were assessed for cell death by flow cytometry following staining for Annexin-V, 7-AAD, CD45, CD34, and CD38. Treatment with APL-030 caused a statistically significant increase in AML cell death in all six samples studied. Three samples exhibited a measurable CD34+/CD38- compartment, enabling evaluation of this leukemic stem cell-enriched population. APL-030 caused a statistically significant increase in cell death in the leukemic stem cell-enriched population in all three samples studied. APL-030 is a novel GCN2 inhibitor that that has shown encouraging efficacy in preclinical studies using both AML cell lines and patient-derived AML samples. Based on these preclinical results, a phase 1/2 study is planned in hematological tumors. Citation Format: Monica Roman-Trufero, Gavin Whitlock, Matthew Fuchter, Maxmila Jeyakumar, Panagiota Chaida, Sereina Annik Herzog, Armin Zebisch, Richard Butt, Holger W. Auner, Nadine Clemo. Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 618.

Abstract 2478: Inhibition of CD39 by AB598 increases extracellular ATP resulting in activation of myeloid cells and T cells to enhance anti-tumor immunity

Abstract Extracellular ATP (eATP) is a potent immunostimulatory signal in the tumor microenvironment (TME), but it is rapidly degraded into AMP and immunosuppressive adenosine. Therefore, ATP metabolism is an attractive anti-tumor target. Several enzymes are involved in the immunosuppressive process used by the TME to convert ATP to AMP and adenosine. Among them, CD39 (gene: ENTPD1), an extracellular ATPase, is a promising target. Analyzing mRNA expression from the TCGA, we show that CD39 is consistently expressed at high levels in comparison to the other extracellular ATP-converting enzymes, ENPP1 and TNAP. We further demonstrate that CD39 has greater enzymatic activity at the slightly acidic physiological pH (pH = 6.8) of the TME than either ENPP1 or TNAP. Flow cytometry profiling of dissociated primary human gastric tumors showed that CD39 was highly expressed on both myeloid and lymphoid cells, including several populations of T cells present in the tumors. This is distinct from findings in healthy primary human PBMCs that show high expression of CD39 on myeloid subsets and relatively few subsets of T cells with high CD39 expression. AB598 is a novel, humanized, Fc-silent anti-CD39 therapeutic antibody that potently binds to CD39 and inhibits its enzymatic activity with sub-nanomolar potency. Treatment of dissociated primary human gastric tumor cells with AB598 resulted in a decrease in the rate of ATP degradation, indicating both the effectiveness of AB598 and the relevance of CD39 as a main contributor to the ATPase activity of the TME. Although eATP is low in healthy tissues, high levels of ATP can be released when cancer cells are treated with immunogenic cell death (ICD)-inducing agents, increasing the eATP in the TME, providing a rationale for the combination of ICD-inducing chemotherapy and AB598. Thus, cancer cells were co-cultured with monocyte-derived dendritic cells (moDCs) and treated with chemotherapy and AB598 to preserve the eATP released by dying cancer cells. The increased expression of CD83 and CD86 on moDCs demonstrated that the combination treatment enhanced the maturation of moDCs, compared to single or no treatment. To further elucidate the anti-tumor effects of the combination treatment, autogenous T cells were introduced into this system. AB598 significantly enhanced T cell activation in the presence of chemotherapy. These data demonstrate that using AB598 results in elevated eATP, which boosts immune cell activation to promote anti-tumor immunity. Citation Format: Ke Jin, Julie Clor, Kaustubh Parashar, Rebecca Fuchs, LC Stetson, Sean Cho, Matthew J. Walters, Ester Fernandez-Salas, Christine E. Bowman. Inhibition of CD39 by AB598 increases extracellular ATP resulting in activation of myeloid cells and T cells to enhance anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2478.

Abstract 36: The discovery of a novel CD19xCD22 dual-targeting CAR for the development of an IPSC-derived cell therapy

Abstract Dual-targeting cell therapies to address CD19 antigen loss in heme malignancies are being explored by Century Therapeutics and others for unmet clinical need in hematological malignancies. CD22 chimeric antigen receptor (CAR) T-cell therapies have shown promising efficacy in relapsed or refractory B-lineage acute lymphoblastic leukemia (B-ALL) alone and in combination with CD19 CAR-T cell therapies (1,2). CD22 is a multi-domain glycoprotein expressed on B-cells. Natural splice variants of CD22 can exclude domains near the N-terminus, allowing for therapeutic resistance when these domains are targeted. We identified novel single domain antibodies (VHH) to CD22 that bind to multiple epitopes on different domains of CD22, including membrane proximal binders. The VHHs were affinity matured to enhance cell-based binding and formatted as CARs in monovalent and tandem, bivalent formats. Our data demonstrates the best efficacy against CD22+ tumor cells with a biparatopic, tandem VHH CAR format. Through additional engineering, bispecific CARs that combined three CD22-binding VHH and the anti-CD19 scFv, FMC63, in a loop CAR configuration were designed and tested. This CD19xCD22 bispecific, CD22 biparatopic CAR was engineered into primary T-cells and demonstrated cytotoxicity activity against CD19 and CD22 positive tumor cells as well as CD19 knockout and CD22 knockout cell lines. CD19xCD22 bispecific primary CAR-T cells were efficacious in controlling tumors in mouse xenograft models. This novel CAR is being tested in IPSC-derived iT and iNK cells for off-the-shelf allogeneic cell therapy to expand patient access beyond CD19 CAR-T cell therapies. Ref: 1. Nat Med. 2018;24(1):20-28. doi: 10.1038/nm.4441. 2. Blood. 2018;132(suppl 1). Abstract 27 Citation Format: Jill M. Carton, John Wheeler, Chris Dower, Liam Campion, Hillary J. Millar, Marilda Beqiri, Barry Morse, Buddha Gurung, Rebecca Genovese, Steven DeLuca, Charles Dominick, Michael Naso, Hy Levitsky. The discovery of a novel CD19xCD22 dual-targeting CAR for the development of an IPSC-derived cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 36.

Abstract 3270: SH3GLB1 is involved with hydrogen peroxide/akt signaling in glioblastoma multiforme cells

Abstract Glioblastoma (GBM), a malignant brain tumor, has poor survival outcomes due to recurrence or drug resistance. In our previous studies, it was found that superoxide dismutase 2 (SOD2) was associated with temozolomide (TMZ) resistance. Moreover, SOD2 can convert O2 •− to H2O2, and H2O2 redox signaling is vital for physiological reactions and promotes tumor progression. We used cell lines, patient primary cells and pharmacological inhibitors/activators to confirm the significance of H2O2 signaling. We used the cultured cells to verify roles of H2O2 signaling in the plasticity of cell fate and did animal experiments to identify the optimal treatment strategy. In the present study, SOD2 was found to be the upstream mediator, and combining SOD inhibitor and TMZ, the cells showed reduced SH3GLB1 and autophagy levels. We then found that SH3GLB1 was regulated by H2O2 through AKT signaling using experiments of regulating redox homeostasis. The change of intracellular H2O2 levels in mitochondria following the treatments is similar to that in cytosol, however, the cell fate of parental or resistant cells was quite diverse. Notably, CCCP, a mitochondrial membrane potential disrupter, increased endogenous H2O2 production, and TMZ-increased SH3GLB1 levels can be reversed by HgCl2, reflecting the aquaporin function. In animal models, a combination of TMZ and 4-hydroxynonenal reduced growth of the SH3GLB1-knockdown resistant cells, along with suppressed autophagy levels. Our results characterizing the mechanism of H2O2-SH3GLB1 axis signaling in acquired TMZ resistance of GBM cells will provide new therapeutic strategies against the disease in the future. Citation Format: Chia-Hung Chien, Kwang-Yu Chang. SH3GLB1 is involved with hydrogen peroxide/akt signaling in glioblastoma multiforme cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3270.

SARS-CoV-2 infection of endothelial cells, dependent on flow-induced ACE2 expression, drives hypercytokinemia in a vascularized microphysiological system.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, has caused nearly 7 million deaths worldwide. Severe cases are marked by an aggressive inflammatory response known as hypercytokinemia, contributing to endothelial damage. Although vaccination has reduced hospitalizations, hypercytokinemia persists in breakthrough infections, emphasizing the need for disease models mimicking this response. Using a 3D microphysiological system (MPS), we explored the vascular role in SARS-CoV-2-induced hypercytokinemia. The vascularized micro-organ (VMO) MPS, consisting of human-derived primary endothelial cells (ECs) and stromal cells within an extracellular matrix, was used to model SARS-CoV-2 infection. A non-replicative pseudotyped virus fused to GFP was employed, allowing visualization of viral entry into human ECs under physiologic flow conditions. Expression of ACE2, TMPRSS2, and AGTR1 was analyzed, and the impact of viral infection on ACE2 expression, vascular inflammation, and vascular morphology was assessed. The VMO platform facilitated the study of COVID-19 vasculature infection, revealing that ACE2 expression increased significantly in direct response to shear stress, thereby enhancing susceptibility to infection by pseudotyped SARS-CoV-2. Infected ECs secreted pro-inflammatory cytokines, including IL-6 along with coagulation factors. Cytokines released by infected cells were able to activate downstream, non-infected EC, providing an amplification mechanism for inflammation and coagulopathy. Our findings highlight the crucial role of vasculature in COVID-19 pathogenesis, emphasizing the significance of flow-induced ACE2 expression and subsequent inflammatory responses. The VMO provides a valuable tool for studying SARS-CoV-2 infection dynamics and evaluating potential therapeutics.

Primary Mouse Aortic Smooth Muscle Cells Exhibit Region- and Sex-Dependent Biological Responses In Vitro.

Despite advancements in elucidating biological mechanisms of cardiovascular remodeling, cardiovascular disease (CVD) remains the leading cause of death worldwide. When stratified by sex, clear differences in CVD prevalence and mortality between males and females emerge. Regional differences in phenotype and biological response of cardiovascular cells are important for localizing the initiation and progression of CVD. Thus, to better understand region and sex differences in CVD presentation, we have focused on characterizing in vitro behaviors of primary vascular smooth muscle cells (VSMCs) from the thoracic and abdominal aorta of male and female mice. VSMC contractility was assessed by traction force microscopy (TFM; single cell) and collagen gel contraction (collective) with and without stimulation by transforming growth factor-beta 1 (TGF-β1) and cell proliferation was assessed by a colorimetric metabolic assay (MTT). Gene expression and TFM analysis revealed region- and sex-dependent behaviors, whereas collagen gel contraction was consistent across sex and aortic region under baseline conditions. Thoracic VSMCs showed a sex-dependent sensitivity to TGF-β1-induced collagen gel contraction (female > male; p = 0.025) and a sex-dependent proliferative response (female > male; p < 0.001) that was not apparent in abdominal VSMCs. Although primary VSMCs exhibit intrinsic region and sex differences in biological responses that may be relevant for CVD presentation, several factors-such as inflammation and sex hormones-were not included in this study. Such factors should be included in future studies of in vitro mechanobiological responses relevant to CVD differences in males and females.

Evolution of nasal and olfactory infection characteristics of SARS-CoV-2 variants.

SARS-CoV-2 infection of the upper airway and the subsequent immune response are early, critical factors in COVID-19 pathogenesis. By studying infection of human biopsies in vitro and in a hamster model in vivo, we demonstrated a transition in nasal tropism from olfactory to respiratory epithelium as the virus evolved. Analyzing each variant revealed that SARS-CoV-2 WA1 or Delta infect a proportion of olfactory neurons in addition to the primary target sustentacular cells. The Delta variant possessed broader cellular invasion capacity into the submucosa, while Omicron displayed enhanced nasal respiratory infection and longer retention in the sinonasal epithelium. The olfactory neuronal infection by WA1 and the subsequent olfactory bulb transport via axon were more pronounced in younger hosts. In addition, the observed viral clearance delay and phagocytic dysfunction in aged olfactory mucosa were accompanied by a decline of phagocytosis-related genes. Further, robust basal stem cell activation contributed to neuroepithelial regeneration and restored ACE2 expression postinfection. Together, our study characterized the nasal tropism of SARS-CoV-2 strains, immune clearance, and regeneration after infection. The shifting characteristics of viral infection at the airway portal provide insight into the variability of COVID-19 clinical features, particularly long COVID, and may suggest differing strategies for early local intervention.

Sensory cells in tunicates: insights into mechanoreceptor evolution.

Tunicates, the sister group of vertebrates, offer a unique perspective for evolutionary developmental studies (Evo-Devo) due to their simple anatomical organization. Moreover, the separation of tunicates from vertebrates predated the vertebrate-specific genome duplications. As adults, they include both sessile and pelagic species, with very limited mobility requirements related mainly to water filtration. In sessile species, larvae exhibit simple swimming behaviors that are required for the selection of a suitable substrate on which to metamorphose. Despite their apparent simplicity, tunicates display a variety of mechanoreceptor structures involving both primary and secondary sensory cells (i.e., coronal sensory cells). This review encapsulates two decades of research on tunicate mechanoreception focusing on the coronal organ's sensory cells as prime candidates for understanding the evolution of vertebrate hair cells of the inner ear and the lateral line organ. The review spans anatomical, cellular and molecular levels emphasizing both similarity and differences between tunicate and vertebrate mechanoreception strategies. The evolutionary significance of mechanoreception is discussed within the broader context of Evo-Devo studies, shedding light on the intricate pathways that have shaped the sensory system in chordates.